The success of blood transfusion often depends on the degree of compatibility between donor and recipient. The degree of compatibility, in turn, is a function of the similarity in Red Blood Cell (RBC) antigen content between donor and recipient. Expression of many RBC antigens in an individual can be predicted from the analysis of their genomic DNA. Therefore, analysis of donor and/or recipient DNA can be used to facilitate blood matching and thus enable proper blood transfusion practice.
Hemolytic reactions are more common in multi-transfused than in singly transfused individuals, not only because of the increased probability of such an event as the number of transfused units increases, but also because of the accumulative nature of the immune response in the recipient. An example of a condition whose treatment includes repeated blood transfusions is Sickle Cell Disease (SCD). From the above follows that a high degree of compatibility with donor blood is often critical for the long-term success of transfusion in SCD patients.
While SCD is more prevalent among individuals of African ancestry, the blood donor population in the USA and other Western countries is largely Caucasian. As a consequence of this disparity, differences in RBC antigens between both racial groups often become responsible for blood transfusion failures in SCD patients.
The genetic variant RHD*DIIIa-CE(4-7)-D, also known as RHD-CE-DS, RHD-CE(4-7)-D, (C)ceS, or r′S, (RHD*r′S henceforth) can be found in up to 5-10% of the African-American population, but is extremely rare in Caucasians. This variant poses a special challenge to blood transfusion because it encodes a rather complex antigen profile, which includes absence of D antigen, altered forms of C (C+W) and e antigens, expression of low-frequency VS antigen, no expression of V antigen, and absence of the high-frequency hrB antigen. Among them, D and C antigens are the clinically most relevant ones.
The antigenic complexity of RHD*r′S correlates with its genetic complexity, which includes a substitution of part of RHD exon 3, RHD exons 4-7, and the intervening introns by their RHCE counterparts, a G>T substitution at position 186 (exon 2), a C>T substitution at position 410 (hybrid exon 3), a C>G substitution at position 733 (exon 5), and a G>T substitution at position 1006 (exon 7). In addition to the changes in the RHD gene, RHD*r′S occurs in cis with RHCE*ceS1006T, an RHCE gene that also encodes substitutions C>G at position 733 (exon 5) and G>T at position 1006 (exon 7).
To add to the antigenic and genetic complexity, knowledge about the molecular basis of RHD*r′S is incomplete. For instance, the precise points of RHCE/RHD recombination have not been reported to date. Furthermore, two types of RHD*r′S variant have been described and named Type 1 and Type 2, which differ not only in their genetic composition but also in their antigen profiles.
Several publications (Westhoff et al., Transfusion (2010), 50: 1303-1311, Pham et al., Transfusion (2009), 49: 495-504, and Tax et al., Transfusion (2002) 42: 6234-6644) have uncovered the genetic similarity between RHD*r′S and other RHD variants, in particular RHD*DIIIa and RHD*DIVa/RHD*DIVa-2 (RHD*DIVa-2 henceforth). A number of molecular methods for the specific detection of RHD*r′S rely on the detection of polymorphisms located in D-CE hybrid exon 3 locus of RHD. These polymorphisms are now known to be shared with variants RHD*DIIIa and RHD*DIVa-2. Consequently, to date, identification of RHD*r′S in a sample by DNA analysis requires detection of hybrid exon 3 polymorphisms and discrimination from RHD*DIIIa and RHD*DIVa-2. This discrimination is clinically relevant since the latter variants encode a different antigen profile, which includes expression of partial D and absence of C+W.
EP1780217 describes the detection of RHD positive haplotypes in D-negative individuals. Silvy et al., British Journal of Haematology, (2012 Dec. 30) doi: 10.1111/bjh. 12179 [Epub ahead of print], describe identification of a polymorphism said to be restricted to the (C)ceS type 1 haplotype. WO2012/171990 describes discrimination of blood type variants in a method making use of polymorphisms in intron 7 of the RHD gene and/or intron 7 of the RHCE gene. EP2471949 describes a method for the identification by molecular techniques of genetic variants that encode no D antigen (D−) and altered C antigen (C+W).
Antibody reagents commonly used to detect C antigen do not discriminate between C+W and C+. Therefore, the phenotype is often reported as C+. In cases where the antibody reagent does discriminate between C+W and C+ but the sample contains a normal RHCE*C allele in trans to a RHD*r′S allele, C+W is obscured by C+, resulting in a C+ phenotype for the sample. Therefore, RHCE*C needs to be tested for and shown absent prior to assignment of a C+W phenotype to a sample. Accordingly, there remains a need for further methods for distinguishing RHD*r′S from RHD*DIIIa and RHD*DIVa-2. The present invention addresses these and other objects.